Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art.
Introduction
Ion channels are a diverse group of proteins that regulate the flow of ions across cellular membranes. In the nervous system, ion channel activity has evolved into a rapid and accurate system for intercellular communication. The electrical excitability characteristics of each neuron is in part determined by the set of channels it expresses. However, cells are also able to regulate the activity of individual channels in response to physiological or developmental events, and there is growing evidence that ion channels can be the site of integration of multiple electrical and biochemical pathways.
In vivo, ion channels appear to be multimeric proteins that are comprised of several distinct gene families, coding for channels with distinct structural and functional properties.
Within a gene family, the potential for heterogeneity arising from the combinatorial assembly of different pore-forming and auxiliary subunits (Greene, et al., 1995). Channel properties can be modulated by second messenger cascades and can directly bind intracellular proteins such as kinases suggesting that this may be an important way to efficiently target the signaling cascade to its effector molecule. The electrical characteristics of each neuron is, in part, determined by the set of ion channels that it expresses. However, cells are also able to regulate the activity of individual channels in response to physiological or developmental events; pore-forming (xcex1) subunits can interact with a variety of intracellular proteins, including auxiliary (xcex2) subunits, cytoskeleton-associated proteins and protein kinases (Greene, et al., 1995). In addition to auxiliary (xcex2) subunits, pore-forming subunits can interact with a variety of intracellular proteins and second messenger molecules themselves including G-proteins, cytoskeleton-associated proteins and protein-kinases (Adelman, et al., 1995).
Several classes of ion channels bind directly, and are regulated by, second messenger molecules such as cyclic nucleotides (Zagotta, et al., 1996; Bruggemann, et al., 1993, and Hoshi, et al., 1995) or Ca+2 (Adelman, et al., 1992; Kohler, et al., 1996). Channels with this property may be key elements in the control of neuronal signaling, as they directly couple biochemical cascades with electrical activity. Cyclic nucleotide-gated channels (CNG) play a distinct role both in visual and olfactory signal transduction; their recent identification in the hippocampus and other regions of the brain, where cAMP and cGMP are known to mediate different forms of synaptic plasticity (Krapivinisky, et al., 1995; Frey, et al., 1993; Bolshakov, et al., 1997; and Arancio, et al., 1995), suggests that CNG-channels may also contribute to the regulation of excitability in central neurons (Kingston, et al., 1996 and Bradley, et al., 1997).
The first structural gene for a K+ channel to be isolated was the gene encoded by the Shaker (Sh) locus in Drosophila melanogaster (Strong, et al., 1993; Papazian, et al., 1987). Its sequence is the prototype of a large and still expanding family of related genes (Kamb, et al., 1987; Warmke, et al., 1994). The properties of a number of well characterized K+ currents, that still await a molecular definition, predicts that other members of this family are yet to be identified (Atkinson, et al., 1991).
Although the initial members of the K+ channel superfamily were cloned by chromosomal localization of alleles responsible for functional defects (Sh, eag and slo from Drosophila; (Papazian, et al., 1987; Kamb, et al., 1987; Warmke, et al., 1991; Atkinson, et al., 1991) or following the purification of a relatively abundant protein such as the cGMP-channel from bovine retina (Liman, et al., 1994), the most widely used strategy for cloning new members of the K+ channel superfamily is by homology to these sequences. Unfortunately, this approach is not well suited for identifying more divergent sequences and potentially new branches in the phylogenetic tree of the K+ channel superfamily. Expression cloning in Xenopus oocytes can circumvent this problem; this implies a pre-existing or readily detectable physiological characterization of the channel.
The present invention provides an isolated nucleic acid encoding a BCNG protein or a portion thereof. The present invention further provides an isolated nucleic acid encoding a BCNG-related protein or a portion thereof. Further, the present invention provides a vector, which comprises cDNA encoding mBCNG-1 (ATCC Designation No. 209781). In addition, the present invention further provides a vector, which comprises cDNA encoding hBCNG-1 (ATCC Designation No. 209827). The present invention also provides an isolated BCNG protein. Further, the present invention also provides an isolated BCNG-related protein.
The present invention additionally provides a composition comprising a nucleic acid encoding a BCNG protein or a portion thereof, or a BCNG-related protein or a portion thereof and a carrier. In addition, the present invention further provides a composition comprising a BCNG protein or a portion thereof, or a BCNG-related protein or portion thereof and a carrier.
Additionally, the present invention provides a nucleic acid probe capable of specifically hybridizing with a nucleic acid encoding a BCNG protein or BCNG-related protein.
The present invention provides a method for identifying a nucleic acid in a sample which encodes a BCNG protein or a BCNG-related protein which comprises: (a) contacting the sample with a nucleic acid probe capable of specifically hybridizing with nucleic acid encoding a BCNG protein or a BCNG-related protein under conditions permissive to the formation of a complex between the nucleic acid probe and the nucleic acid encoding the BCNG protein or the BCNG-related protein in the sample; (b) determining the amount of complex formed in step (a); and (c) comparing the amount of complex determined in step (b) with the amount of complex formed using an arbitrary sequence, a greater amount of complex formed with the BCNG-specific probe indicating the presence of a nucleic acid encoding a BCNG protein or a BCNG-related protein in the sample.
Further, the present invention provides a method for testing whether a compound affects the expression of a BCNG protein or a BCNG-related protein which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound; (b) determining the amount of expression of BCNG protein or BCNG-related protein in the sample; and (c) comparing the amount of BCNG protein or BCNG-related protein expression determined in step (b) with the amount determined in the absence of the compound.
In addition, the present invention further provides a method for identifying a compound capable of interacting with a BCNG protein or a BCNG-related protein which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound under conditions permissive to formation of a complex between the compound and the BCNG protein or the BCNG-related protein; (b) determining the amount of complex formed between the compound and the BCNG protein or the BCNG-related protein; (c) comparing the amount of complex formed in step (b) with the amount formed in the absence of the compound, a greater amount of complex formed in the presence of the compound indicating the presence of a compound capable of interacting with a BCNG protein or a BCNG-related protein.
Also, the present invention provides a method for identifing a compound capable of modulating BCNG protein or BCNG-related protein activity which comprises: (a) contacting a sample which expresses a BCNG protein or a BCNG-related protein with a compound; (b) determining the amount of activity of the BCNG protein or BCNG-related protein in the sample; and (c) comparing the amount of activity of the BCNG protein or the BCNG-related protein determined in step (b) with the amount determined in the absence of the compound, an increase or decrease in activity indicating the presence of a compound capable of modulating the activity of the BCNG protein or the BCNG-related protein.
Further, the present invention also provides a method of treating a condition in a subject which comprises administering to the subject an amount of the provided compound, effective to treat the condition.
Finally, the present invention provides a pharmaceutical composition which comprises the provided compound and a pharmaceutically acceptable carrier.